The overall objective of the proposal is to identify hair cell/efferent transmitters/modulators at the periphery, the receptors mediating their actions, and the basic biochemical events occurring in hair-cell synaptic transmission as modulated by these transmitters that initiate afferent signaling in octavolateralis systems. In this new investigation, we will focus on the role of serotonin as an efferent transmitter within the auditory organ of Corti and as a receptoneural transmitter in vestibular hair cells. We will investigate norepinephrine-containing adrenergic efferents constituting a newly-recognized neural system operating within the organ of Corti and examine the newly-discovered efferent neuromodulator PACAP as to its sites of action and receptors. Molecular interactions of hair cell voltage-gated calcium channels (VGCC) with synaptic proteins and with scaffolding proteins will be investigated. In Aim 1, we will continue the identification of neurotransmitters/neuromodulators in hair cells and inner-ear sensory epithelia, with femtomole-sensitive, high-resolution, high-performance liquid chromatography. Enzymes of synthesis and transporters for the transmitters will be analyzed with molecular and immunohistochemical protocols. Release, a criterion of neurotransmitter identification, will be investigated in response to sound stimulus in vivo. In Aim 2, we will continue to characterize hair-cell voltage-gated calcium channels that give rise to the calcium-induced exocytotic signal via surface plasmon resonance and pull-down binding assays, examining protein-protein interactions. In Aim 3, the corresponding neurotransmitter receptors - membrane-bound proteins that receive and respond to transmitters - will be characterized as to their molecular functional domains and their neural and hair-cell immunolocalizations, defining mechanisms for afferent/efferent signaling in the acoustico-lateralis periphery. These approaches, utilizing microbiochemical methods, should result in continued, detailed elucidation of structure and molecular function of peripheral neurotransmitter systems of hearing and balance, pointing the way to development of biochemical and genetic therapies for transmitter-related hearing loss, vertigo, and tinnitus. The possibility of biochemical/pharmacologic manipulation of afferent signaling in auditory/vestibular deficits such as deafness, tinnitus, and dizziness is completely dependent upon our knowledge of basic elements of hair-cell receptoneural transmission, as modulated by efferent neurotransmission. Recent advancements in the identification of new neurotransmitters/neuromodulators in hair cell-containing sensory epithelia point to continuing relevance and need for this kind of investigation. Identification of the presently unknown outer hair cell transmitter, considered in the proposal, has implications for peripherally-derived tinnitus. Serotonergic and adrenergic efferents are implicated in modulating otoacoustic emissions and are likely to be involved in determining the set point for the outer hair cells. Native neuroprotective mechanisms for the type I afferent dendrites is an issue addressed in the present proposal for the neuropeptide PACAP, important in excitotoxicity and noise-induced deafness. Characterization of the interactions of voltage-gated calcium channels with synaptic complex proteins has implications for providing the necessary elements to reverse deafness, such as DFNB9, caused by mutations in the synaptic complex protein, otoferlin.